Brefeldin A (BFA) is a fungal metabolite with a broad range of biological activities, including the anti-fungal and anti-tumor activities. In the past few years there has been tremendous interest in its effect on cells, since it has been found to have the unique ability to interfere with vesicular trafficking. Among its effects, it has the ability to disperse the Golgi complex, returning at least some of its components to the endoplasmic reticulum (ER). BFA also prevents newly synthesized proteins destined for the cell surface from leaving the ER. To better understand the mechanism of BFA action we conjugated it to two fluorescent dyes and studied its intracellular localization. Both conjugates maintained their biological activity, and both specifically localized to the ER and Golgi complex in live or aldehyde-fixed cells. The interaction with intracellular membranes is probably due to interaction with lipids as it is abolished by detergent extraction of lipids. These conjugates are the first dyes that bind the ER and Golgi complex without binding other prominent membrane bound compartments and should prove useful as probes for the these organelles in living cells. Their ability to stain the ER of fixed cells will be of great use as markers of the ER in cytochemical studies. Finally, the interaction of BFA with the ER and GC membranes might be critical to its biological activity.